DNA-RNA and RNA-RNA hybridization are important to many aspects of nucleic acid function including DNA replication, transcription, and translation. Hybridization is also central to a variety of technologies that either detect a particular nucleic acid or alter its expression. Antisense nucleotides, for example, disrupt gene expression by hybridizing to target RNA, thereby interfering with RNA splicing, transcription, translation, and replication. Antisense DNA has the added feature that DNA-RNA hybrids serve as a substrate for digestion by ribonuclease H, an activity that is present in most cell types. Antisense molecules can be delivered into cells, as is the case for oligodeoxynucleotides (ODNs), or they can be expressed from endogenous genes as RNA molecules. The FDA recently approved an antisense drug, Vitravene® (for treatment of cytomegalovirus retinitis), reflecting that antisense has therapeutic utility.
A recently identified application for antisense ODNs is targeted disruption of expression of genes having unknown function. The nucleotide sequence of all genes of many species, including human, is becoming available to the research community, but the function of only a fraction is known. The widespread application of gene chip arrays will allow hypotheses to be developed about gene circuitry, and antisense ODNs will make it possible to test these hypotheses by down regulation of specific genes singly and in combination. The use of antisense ODNs is expected to increase as genomic nucleotide sequences become available for more organisms and as more pharmaceutical companies use this information to seek new drugs.
Widespread use of antisense nucleic acids nonetheless faces a serious obstacle. Extensive RNA structure that impedes antisense-RNA hybridization makes it difficult to identify favorable sites in a target RNA for antisense binding. Since equilibrium is unlikely to be achieved inside cells, understanding hybridization is likely to require an accurate description of hybridization rate. The challenge is substantial because hybridization rate can be very fast even though RNAs are expected to contain considerable secondary structure. For example, in Escherichia coli regulatory antisense RNAs form stable complexes with their target RNA at second order association rates that are close to the upper limit for unstructured RNA association (106 M−1sec−1; Persson et al., 1988; Porschke and Eigen, 1971; Tomizawa, 1984)).
Previous efforts to describe hybridization between oligonucleotides and target RNA fall roughly into two groups. In one, predictions of RNA secondary structure were used to identify regions likely to be single stranded and presumably accessible for hybridization (Christofferson et al., 1994; Patzel et al., 1999). Correlation with oligonucleotide hybridization showed considerable scatter, and we now know that single-stranded regions, identified by nucleases, do not correspond to those that hybridize most readily (see FIG. 3 in Birikh et al., 1997). In the second approach, overall energy gain due to hybrid formation was calculated (Stull et al., 1992). This method, which focuses on equilibrium yield of hybrids, provided a poor correlation between energy gain and hybridization of antisense oligonucleotides to RNA (Stull et al., 1992). More recently, Mathews et al. analyzed two experiments (Mathews et al. 1999a). The data from one correlated with the equivalent of ΔGd (FIG. 1) while the data of the other correlated with the equivalent of ΔGh (FIG. 1). Thus, no general treatment has been available, making it necessary to identify favorable sites for antisense attack of mRNA experimentally (Birikh et al., 1997; Branch, 1998).
A current procedure used to identify such favorable sites involves construction of random sequence ODN libraries, expression and purification of target mRNA, hybridization of library ODNs to target RNA, cleavage of hybrids with RNase H, gel electrophoresis of cleavage products to determine their sizes, and primer extension to accurately determine the cleavage sites. For most laboratories interested in gene function, this procedure for identifying favorable sites for hybridization is a project unto itself A need exists, therefore, for accurate but less time- and labor-intensive methods for identifying favorable sites for hybridization.